Dna vaccine, method of inducing the immune response, method of immunisation, antibodies specifically recognising the h5 haemagglutinin of an influenza virus and use of the dna vaccine

ABSTRACT

The object of the invention is a DNA vaccine, method of inducing the immune response, antibodies specifically recognising the haemagglutinin H5 of an influenza virus and application of the DNA vaccine. According to the invention, one or two-fold immunisation of hens with DNA vaccine containing a cDNA encoding the modified H5 haemagglutinin HA protein, i.e. with the deletion of the cleavage site between HA subunits (this provides for greater safety of the vaccines). Moreover, the encoding region of the HA is modified in such a way that protein production in the bird cells should achieve maximal yield. The main modification is codon optimisation for the hens and deletion of the site of proteolytic cleavage between subunits HA1 and HA2.

The subject of the invention is a DNA vaccine, method of inducing theimmune response, method of immunisation, antibodies specificallyrecognising the haemagglutinin of the H5 subtype of an influenza virusand use of the DNA vaccine. According to the invention, one or two-timeimmunisation of hens with a DNA vaccine containing the cDNA encoding themodified H5 haemagglutinin (HA) protein, i.e. with the deletion of theproteolytic cleavage site between HA subunits (this provides for greatersafety of the vaccines and the expression of a “super antigen” in theform of a long, non-processed polypeptide). Moreover, the encodingregion of the HA is modified in such a way that protein production inthe bird cells should achieve maximal yield. The main modification iscodon optimisation for the hens and deletion of the proteolysissensitive region of HA.

Over the recent years, exceptional attention has been focused on theinfluenza virus, especially the highly pathogenic type of the avianinfluenza virus (HPAI). “Bird flu” is a serious and highly contagiousdisease of poultry and other breeding birds. Actually, many strains ofthe avian influenza virus are circulating, and some of them could alsopass to mammals, in particular pigs and people. The level of threat foranimal health and the public health caused by different strains ofinfluenza viruses in the bird population is highly variable and, to acertain extent, unpredictable because of frequent point mutations andthe possibility of the replacement of RNA segments between differentstrains. Infection with some strains of influenza viruses coming frombirds might be the source of illness amongst domestic fowl. Thecollaborative research of epidemiologists and ornithologists led to theconclusion that it is not the migration of birds, but ratherincompatibility with the obligatory sanitary-veterinary regulations ofpoultry breeding and trade that is responsible for the transfer of avianinfluenza. At present, it seems that the most effective method ofcounteraction for the transfer of avian influenza to people (from whichan influenza pandemic could be an effect) and minimisation of potentiallosses in the poultry industry is immunisation of a breeding flock atrisk against avian influenza viruses, especially against the highlypathogenic H5N1 strains. It is worthwhile adding that vaccination oflaying hens would prevent the possible spread of the virus through theegg distribution chain and allow for the protection of chicks in theirearly lifespan through maternal antibodies present in the yolk of eggs.

Two main types of conventional vaccines against influenza are acceptedfor use on people. The first one, the most frequently used, is theclassical inactivated vaccine comprising inactivated virions. In orderto obtain these kinds of vaccines, the viruses must be multiplied and,after purification, subjected to chemical or physical inactivation. Thesecond one is a live attenuated vaccine (e.g. FluMist), which consistsof reassortants between a vaccine virus and a temperature sensitivevirus, so-called cold adapted (And/Ann Arbor/6/60), which can multiplyexclusively in temperatures up to 33° C.

At present, the conventional vaccines used are trivalent. Before everyinfluenza season, WHO decides what strains of viruses will be the sourceof the recommended vaccine. Such seasonal vaccines contains threeinfluenza viruses: one influenza virus type A subtype H3N2, one type Asubtype H1N1 and one influenza type B of the virus strain. Theeffectiveness of inactivated vaccines is about 30%-90% depending on ageand medical conditions. Live attenuated vaccines are characterised by ahigher effectiveness and a broader immune response (humoral, cellular,MALT).

Actually, the availability of technology for mass production of viralparticles is essential for the preparation of conventional vaccines. Themost classical method used is propagation/multiplication of influenzaviruses in embryonated hen eggs. After multiplication, the virus isisolated along with the amniotic fluid. This technique has somedisadvantages. First of all, it is lengthy and time-consuming. Moreover,during repeated passages in chicken embryos, the glycoproteins of thevaccine virus, in particular in the HA coding gene, undergo gradualmutations which may lead to the altered antigenic profile of thevaccine. Obviously, vaccines received by this method are not suited forpeople allergic to egg products.

Another method of large-scale production is multiplying influenzaviruses in cell cultures such as well characterised and accredited celllines like Vera or MDCK. In comparison with breeding in embryos, theproblem of the mutation of the genes of glycoproteins and contaminationwith allergenic chicken proteins is eliminated. However, other problemsarise due to the adherent kind of cell culture, resulting in thenecessity of applying micro-carrier bioreactors of a capacity reaching10,000 litres. This means heavy costs and time-consuming production (˜6months from WHO recommendation to pharmaceutical product). Thus, analternative to conventional vaccines is still missing.

Production of a new generation of vaccines should be fast, safe, simple,cheap and easily applicable. Such features are ascribed to subunitvaccines consisting of strong, closely defined antigens of the virus ortheir most immunogenic fragments. Elements of subunit vaccines might beisolated directly from pathogenic organisms, but this approach requires,once again, pathogen production on a large scale, which is dangerous andexpensive. Applying the technology of the recombined DNA offers anattractive method of attaining immunogenic subunits. This technologycombines the benefits of subunit vaccines with the possibility ofobtaining their components in a non-pathogenic expression system, e.g.bacterial, yeast, mammalian or plant cells. This way of antigenproduction is fast, cheap, safe and highly efficient.

The next group of new generation vaccines are DNA vaccines (geneticvaccines), which are in fact regarded as third generation vaccines. Forthe first time in the early 1990s, DNA vaccines were used forimmunisation of animals. Since that time, the DNA vaccine concept wastested against various pathogens. The effectiveness of DNA immunisationwas shown in models of the following illnesses and pathogens: influenzatype B, HBV, malaria, tuberculosis, SIV viruses and HIV type 1 anddifferent cancers. Importantly, DNA vaccines may affect not only humoralbut also cellular immunity. Moreover, two other advantages of thesevaccines seem to be very important: (a) possibility of rapid design andconstruction of the vaccine, which allows for the avoidance of thelong-term and elaborate procedure of protein expression in selectedsystems and their purification; and (b) attainment of an antigen of anidentical structure with the version arising during infection, i.e.containing post-translational modification typical for the host, sinceprotein production is occurring in situ, as a result of the DNAexpression in immunised animal cells. It's quite easy to increase theimmunogenicity of DNA vaccine rationalisation or adaptation of theencoding sequence to the antigenic sequence optimised set of codons forthe given host, e.g. mouse, human.

The precise mechanism of action of genetic vaccines has been recognisedrather recently. It is postulated that proteins synthesised in the hostas a result of the expression of genes of pathogens engineered in theDNA vaccine “mimic” proteins produced in the course of natural pathogeninfection. Briefly, the DNA plasmid is delivered to the skin or muscleby one of several delivery methods. The plasmid enters the nucleus ofthe transfected local cells, including resident antigen presenting cells(APCs). The expression of plasmid-encoded genes results in generation ofantigenic proteins that are converted to the peptide string by theintracellular proteolytic complexes. These antigens can become thesubject of immune surveillance in the context of both majorhistocompatibility complex (MHC) class I and class II molecules of APCsin the vaccinated organism. In draining lymph nodes, antigen-loaded APCs‘present’ antigenic peptide-MHC complexes in combination with signallingcostimulatory molecules to naïve T cells. This interaction results ininitiation of an immune response, such as activation and expanding of Tcells and activation of B cells and the antibody production process. Inthis way, both humoral and cellular immune responses are triggered. Thedetails concerning current research on genetic vaccines may be found ina few review articles (Dhama et al., 2008; Kutzler and Weiner, 2008)

In May 2006, Vical Incorporated (http://www.vical.com) announced theproduction of a trivalent vaccine containing the DNA sequence encodingH5 antigen, nucleoprotein (NP) and M2 protein. The vaccine isadministrated with the company's patented adjuvant, Vaxfectin. It wasshown that 100% of immunised mice and ferrets are protected againstinfection from the H5N1 flu virus. The vaccine also provides a highlevel of protection against infection with other strains of influenzaviruses.

The basic mechanisms of the immune response of birds are similar to theresponse of mammals. Birds also have an ability to induce the Th1 andTh2 type of immunological response and similar path of their induction.However, the type and the location of the early immune response to theDNA vaccine can be different in birds because of the presence of otherlymphatic bodies. The frequently used promoter of CMV (humancytomegalovirus) is sufficiently active in bird cells in order toprovide efficient expression of genes under its control. The typicaladministration routes of the vaccine are intramuscular injection andsubcutaneous, and the first one seems to be the most effective. DNAvaccines might be injected into birds with or without any adjuvants.Some biological adjutants such as IL-2, IL-8, IL-6 and the γ interferon(IFNγ) were tested. Published data concerning the immunisation ofpoultry with DNA vaccines has shown positive effects in the form of thecellular, humoral or protective response. What's more, with birds inwhich the presence of specific antibodies wasn't demonstrated, aprotective response was detected, which would suggest the appearance ofa cellular rather than humoral response. Moreover, it has been recentlydemonstrated that immunisation of birds with the DNA vaccine encodingchicken IL-2 enhances the immunological response to the IBDV DNA vaccine(Park et al., 2009).

DNA vaccines encoding the HA protein from different strains of avianinfluenza viruses were also tested in poultry. In many cases, theauthors didn't state the presence of anti-HA antibodies, butdemonstrated effective protection against infection with lethal doses ofthe virus. Different ways of vaccine administration were tested, and themost effective was by using the gene gun.

Here are some examples of publications describing the use of the cDNAencoding HA to poultry immunisation: (Fynan et al., 1993; Fynan et al.,1993 b; Robinson et al., 1993; Kodihalli et al., 2000; Rao et al., 2008;Swayne, 2009).

The WO2008145129 (published 2008 Dec. 4) and US20100160421 (published2010 Jun. 24) inventions concern vaccines and the use of the naked DNAand/or RNA molecule encoding haemagglutinin (HA) [and, optionally,encoding neuraminidase (NA) and/or matrix protein (M) and/or thenucleoprotein (NP)] from pandemic influenza as a vaccine componentagainst present day and future (coming) H1, H2, H3, H5, H7, N1, N2, N3containing influenza A infections in humans and/or swine. The influenzaviruses mentioned in the invention included: the 1918 H1N1, the 1957H2N2, the 1968 H3N2 influenza A virus, the high pathogenic bird pandemicATV strain (A/buzzard/Denmark/6370/06(H5N1)), the 2001 H5N7 lowpathogenic avian influenza virus (ATV) strain(A/Mallard/Denmark/64650/03(H5N7)), the March 2006 Denmark H5N1 highpathogenic AIV strain (A/buzzard/Denmark/6370/06(H5N1)), the 2008(A/duck/Denmark/53-147-8/08 (H7N1)), the 2004(A/widegeon/Denmark/66174/G18/04 (H2N3)). If the vaccine components areused as DNA or RNA vaccines with or without the corresponding protein,the codons can optionally be “humanised” using preferred codons fromhighly expressed mammalian genes, and the administration of this DNAvaccine can be by saline or buffered saline injection of naked DNA orRNA or injection of a DNA plasmid or linear gene expressing DNAfragments coupled to particles. Addition of the matrix protein (M)and/or the nucleoprotein (NP) as protein or DNA from the 1918 influenzastrain is also disclosed.

In the invention US20090291472, the codon-optimised nucleic acidsencoding influenza polypeptides and uses of nucleic acids andpolypeptides for inducing immune responses are described.

The invention WO2009092038 (published 2009 Jul. 23) concerns a DNAvaccine against the influenza virus and its use. Sustained outbreaks ofhighly pathogenic avian influenza (HPAI) H5N1 in avian species increasethe risk of reassortment and adaptation to humans. The ability to stopits spread in birds would reduce this threat and help maintain thecapacity for egg-based vaccine production. While vaccines offer thepotential to control avian disease, a major concern of current vaccinesis their inability to protect against evolving avian influenza viruses.DNA vaccines encoding haemagglutinin (HA) proteins from different HPAIH5N1 serotypes protect against the homologous and heterologous HPAI H5N1strain challenge in animals. These vaccines elicit antibodies thatneutralise multiple serotypes of HPAI H5N1 when given in combinationscontaining up to 10 HAs. The response is dose-dependent. The breadth ofprotection is determined by the choice of the influenza virus HA in thevaccine. Monovalent and trivalent HA immunogens and/or vaccinesconferred complete protection in mice against a lethal H5N1A/Vietnam/1203/2004 challenge 68 weeks after vaccination. In chickens,complete protection was conferred against heterologous strains of HPAIH5N 1 after vaccination with a trivalent H5 serotype DNA vaccine withdoses as low as 5 μg DNA given twice either by intramuscular needleinjection or with a needle-free device.

The invention EP2023952 (published 2009 Feb. 18) providespolynucleotides and polypeptides capable of enhancing the immuneresponse of a human in need of protection against influenza virusinfection by administering in vivo, into the tissue of the human, atleast one polynucleotide comprising one or more regions of nucleic acidencoding an influenza protein or a fragment, variant or derivativethereof, or at least one polypeptide encoded therefrom. The presentinvention also relates to identifying and preparing influenza virusepitopes and to polynucleotides and polypeptides comprising suchinfluenza virus epitopes. The present invention also relates tocompositions and methods of use in the prevention and treatment ofinfluenza virus infection.

Despite intensive research and numerous published data, the state of theart still isn't sufficient for creating an economical vaccine forpoultry that would be simple in application and, at the same time, wouldensure effective protection and the possibility of distinguishing hensimmunised from ones infected with a virus. Unexpectedly, it turned outthat the suggested solution, according to the invention, had a chance tofill this gap.

The aims of the invention are: creating a vaccine containing themodified cDNA encoding HA protein of the H5 serotype, drawing up amethod of inducing the immune response, as well as obtaining antibodiesspecifically recognising the H5 HA and using the DNA vaccine forobtaining antibodies recognising the H5 HA of the influenza virus.Realisation of the defined objective and solving the problems describedin the state of the art with ensuring the effective protection andpossibility of distinguishing between chickens immunised against andinfected with the virus with simultaneous simple administration wereachieved in this invention.

According to the invention, a one-dose or a two-dose immunisation ofchickens with a vaccine containing DNA encoding the modified H5 HAprotein with the deletion of the proteolytic cleavage site between HAsubunits is proposed. This modification allows for expression of allknown and prospective HA in a single continuous polypeptide not dividedinto separate domains. This may have a bearing on conformation of theantigen and hence exposition to the immune system, differing favourablyfrom what has been reported in literature. Moreover, application of themodified encoding region is proposed in order to provide a high level ofantigen protein production in the cells of vaccinated birds. Codonsaltered to the codons preferred by domestic chickens is an importantmodification. By applying the solution according to this invention, animmune response of the humoral type was already obtained (production ofthe specific antibodies) in immunised poultry after one- or two-foldimmunisation. Sera taken from immunised birds provided positive resultsin the inhibition test (HI), which lets us assume that the antibodiescould neutralise the flu virus and protect from infection.

Indeed, the challenge experiment confirmed the protective activity ofthe DNA vaccine that was administered two times on day 7 and 21 at adose of 125 μg of plasmid DNA in a PBS solution with the transfectionreagent Lipofectin. The immunised birds were protected against influenzavirus infection, and shedding of the viruses was not observed.

Strengthening of the immune response by additional administration of theplasmid ensuring the cDNA expression of chicken interleukin 2 wasobserved in some cases.

The subject of the invention is a DNA vaccine containing a modified cDNAencoding the haemagglutinin H5, characterised by the presence of thedeletion of the region encoding amino acids in the site of theproteolytic cleavage between HA1 and HA2 units and by optimally alteredcodons for hens in order to provide the maximum yield of H5 protein inbird cells after immunisation.

Preferably, when the vaccine contains the sequence determined by SEQ. IDNo. 2.

Preferably, when the deletion is in a range between 15 and 21 base pairsencoding amino acids of the cleavage site between HA1 and HA2 units,preferably 18 base pairs.

The next subject of the invention is a method of induction of thehumoral immune response in which specific antibodies recognisinghaemagglutinin H5 protein are produced, characterised by that the DNAvaccine described above is applied.

Preferably, when the immune response is enhanced by providing a plasmidensuring the cDNA expression of chicken interleukin 2 determined by thesequence SEQ. ID No. 3.

Preferably, when the first vaccine dose is administered up to the14^(th) day after hatching.

Preferably, when the manner of preparation of expression plasmids andsamples for the immunisation includes lipid or macromolecular carriers.

The next subject of the invention is a method of immunisation,characterised in that, at least one dose of the DNA vaccine describedabove is applied.

Preferably, the first dose of the vaccine is DNA vaccine described inclaims 1 to 3 and the second dose of the vaccine is the DNA vaccine orthe antigenic protein, HA.

The next subject of the invention are antibodies specificallyrecognising the haemagglutinin 1-15 of an influenza virus, characterisedby the fact that they are obtained by using the vaccine specified above.

Preferably, when antibodies are active or inactive in the test ofhaemagglutination inhibition using the H5 antigen of an influenza virus.

Preferably, when antibodies are able or unable to neutralise aninfluenza virus.

In order to better characterise the invention, it is presented in thefollowing figures:

FIG. 1 shows the presence of anti-HA antibodies in the sera of immunisedchickens. The antibodies were detected in the sera of 33-day-oldchickens by dot blot. 100 ng of the HA protein (A/Bar-headedGoose/Qinghai/12/05; Immune Technology) was used as a control antigenthat was spotted on the membrane directly. Each piece of the membranecontaining the control antigen was incubated with the sera of chickensof the designated group (B1-B11). The chicken antibodies were nextdetected with appropriate secondary antibodies. K(+)-positive control(HI AIV H5-positive) from the National Veterinary Research Institute inPulawy. The numbers of groups (B1-B11) are given above the correspondingresults of the test; descriptions of the groups are in scheme A.

FIG. 2 shows the presence of anti-HA antibodies detected by ELISA assayin the sera of individual chickens from the indicated groups on twoindicated dates (day 18 and day 33). The numbers of the groups areexplained in scheme A. K(+)-positive serum from chickens immunised witha sample containing the membrane fraction of recombined baculovirusAcMNPV carrying the full length HA gene.

FIG. 3 shows the results of the HI test of selected chickens immunisedaccording to scheme A. K(+)-positive control (HI anti-serum for AI H5N2;GD Deventer, Netherlands).

FIG. 4 shows the comparison between the response to immunisation withDNA with optimised codons (HA-opt) and with non-optimised codons(HA-nopt). A—results of the dot blot showing anti-HA antibodies in thesera pooled from all chickens of examined groups. B—results of the HItest of individual chickens (indicated by numbers) from both groups;K(+) positive control; the numbers under the bars indicate the highestserum dilutions providing a positive response.

FIG. 5 shows the results of the ELISA test detecting anti-HA IgY in thesera of vaccinated chickens. Samples were collected two weeks after thefirst immunisation (day 20) and three weeks after the secondimmunisation (day 42). The data is shown as mean titers with the SD ofeach group (optical density at 450 nm). *group received one dose, **wildtype HA sequence, ***immunisation without a liposomal carrier

FIG. 6 shows the results of the Haemagglutination Inhibition testdetecting anti-HA antibodies in the serum of vaccinated chickens.Samples were collected three weeks after the second immunisation (day42). Sera without detectable HI were assigned a titer of 0. The data isshown as mean titers with the SD of each group (log₂ from the reciprocalof the highest dilution providing positive result). *group received onedose, **non-optimised HA sequence, ***immunisation without a liposomalcarrier

FIG. 7 shows the results of the avian influenza virus (AIV) challengeexperiment with a homologus strain 3 weeks (A) and 8 weeks (B) afterfinal vaccination, and with a heterologus strain (from a different clad)3 weeks after the final vaccination (C). The data is shown as % of thesurvival ratio in the respective groups (ck—control chickens,tc—transmission chickens, vc—vaccinated chickens).

In order to better understand the invention, examples are listed below.

EXAMPLES Example I Preparation of the Expression Plasmids and VaccineDosages for Immunisation

The cDNA carrying the open coding frame of the full-lengthhaemagglutinin were obtained in the reverse transcription andamplification reaction (RT-PCR) using as a template the RNA of thePolish strain of influenza virus H5N1 (A/swan/Poland/305-135V08/2006);EpiFluDatabase Acc. No. EP1156789; http://platform.gisaid.org;(Gromadzka et al., 2008). The nucleotide sequence is presented as SEQ.ID No. 1. Next, cDNA with deletion of the 18 nucleotides encoding aminoacids 341-346 (RRRKKR at the site of proteolytic cleavage betweensubunits HA1 and HA2) was synthesised. Additionally, the sequence wasoptimised for the domestic chicken (Gallus gallus) codon bias. Thesequence is 100% identical at the amino acid level and 76% at nucleotidelevel compared with the wild type sequence of haemagglutinin (includingthe 18 nucleotide deletion described above) of the Polish H5N1 strain. AcDNA of 1689 by length coding HA protein corresponding to 1-568 aminoacids of HA (with the deletion between the 341-346 aa position) wascloned into the pCI (Promega) vector (restriction sites MluI and SalI)resulting in a K3 plasmid construct. The sequencing of the recombinantplasmid confirmed the correct sequence of the cloned cDNA coding HA, andthe sequence is presented as SEQ. ID No. 2. In a similar way, the pIL2plasmid carrying cDNA of chicken interleukin 2 (chIL-2) was prepared.The IL-2 cDNA was retrieved from the U.D. chick EST database[http://www.chickest.udel.edu] clone number pat.pk0036.g8 (GenBankAF017645). The sequence of chIL-2 is presented as SEQ. ID No. 3.

The obtained recombinant plasmids were transformed into bacterial cells,Escherichia coli DH5α. The recombinant plasmid DNA was isolated andpurified using a NoEndo Jetstar 2.0 Plasmid Giga Kit (Genomed). Theconcentration and purity of the DNAs were estimatedspectrophotometrically at OD₂₆₀ and by separation in agarose gels.Vaccine doses were prepared by mixing DNA with a Lipofectin transfectionreagent (Invitrogen) solution prepared according to the manufacture'sprocedure. The plasmids were mixed with the prepared solution ofLipofectin in a 6:1 ratio (μg DNA: ∥l Lipofectin) or 12:1 for a mixtureof one plasmid or two kinds of plasmids, respectively.

Example II Subcutaneous Immunisation of Chickens and Collection ofSamples for Analysis

Chickens were immunised according to scheme A.

Scheme A of the experiment of subcutaneous immunisation of broilerchickens Priming immunisation Boosting immunisation Group (on day 3) (onday 17) No. of No. Formulation Dose Dose chickens B1 K3 250 μg 250 μg 7B2 K3 125 μg 125 μg 7 B3 K3 + IL2 125 μg + 125 μg 125 μg + 125 μg 7 B4K3 62.5 μg  62.5 μg  7 B5 K3 + IL2 62.5 μg + 125 μg   62 μg + 125 μg 7B6 K3 31.25 μg   31.25 μg   7 B7 K3 + IL2 31.25 μg + 125 μg   31.25 μg +125 μg   7 B8 pCi 250 μg 250 μg 7 B9 IL2 125 μg 125 μg 7 B10 lipofectin— — 7 B11 K3 (-lipofectin) 250 μg 250 μg 7

Immunisation according scheme A. Vaccines were administered subcutaneousin the neck on day 3 and 17 of life. Different doses of DNA vaccineswere administered: 250, 125, 62.5 and 31 μg; all in 400 μl volume. Inaddition, each dose was tested in two variants, one with plasmidencoding interleukin 2 and the second one without this plasmid. Asnegative controls, the groups were injected with an empty vector (250ug) or chIL-2 plasmid (125 μg) or K3 plasmid without Lipofectin or withLipofectin alone (without DNA). Blood samples were taken from the wingvein on day 16 and 33 of life. The blood was coagulated at roomtemperature over 2 h then moved to 4° C. Blood clots were centrifuged at5,000×g at 4° C. for 10 minutes. The sera prepared in such a way werekept at −20° C.

Example III Analysis of Immunological Response by Dot Blot

The presence of specific anti-HA antibodies in sera collected fromimmunised chickens were detected by dot blots. Samples of 100 ng of HAprotein (A/Bar-headed Goose/Quinghai/12/05; Immune Technology) werespotted onto a nitrocellulose membrane. Equal volumes of sera derivedfrom individual chickens of the same group were pooled and diluted 1:200and incubated for 1 hour with a nitrocellulose membrane carrying thespotted antigen (HA protein). Secondary antibodies, anti-chicken IgY-AP,were used, and for their detection, an alkaline phosphate substrate(Roche) was applied. The results indicated the presence of the specificanti-HA antibodies in chickens immunised with K3 and K3+chIL-2. ThechIL-2 seemed to enhance the humoral response, but only in the case of alow dose DNA vaccine (Groups B4-B7) that was 31.2-62.5 μg of K3 plasmid.

Example IV Analysis of Immunological Response by ELISA Assay

The ELISA assay was used to evaluate the presence of anti-HA antibodies.The plates (MaxiSorp, Nunc) were coated with a control recombinantantigen (A/Bar-headed Goose/Quinghai/12/05; Immune Technology) at aconcentration of 3μg/ml in a PBS buffer. The plates without coating byantigens were used to estimate the nonspecific background. The coatingwas performed over night at 4° C. The sera were diluted 1:100 or 1:50 inPBSS (PBS+NaCl) and, for detection, mouse anti-chicken IgY (γ-chainspecific) as the secondary antibodies. The goat anti-mouse IgG-HRP wasthen used. As a substrate for horseradish peroxidase HRP, a TMBchromogen substrate solution (Sigma) was used. After 30 min. incubationwith TMB at room temperature, the reaction was stopped by 0.5 M H₂SO₄.The sample was denoted as positive when its absorbance value measured atOD₄₅₀ was two times higher than the arithmetic mean of the controlgroups.

As shown in FIG. 2, in the groups immunised with the K3 plasmid, astrong immunological response was detected. The response was directlyproportional to the dose of administered plasmid DNA. The results werein agreement with the ones obtained in dot blots (FIG. 1). Moreover,positive signals were also observed in the samples collected earlier,namely 14 days after the first immunisation, e.g. on day 18 of thechicken's life. However, the titer was rather low, and the specificantibodies were only detected in some of the immunised chickens. 100% ofthe chickens of groups immunised with the highest dose of K3 plasmid DNAshowed a high level of specific antibodies in the sera from the secondterm of blood collection (day 33).

Analysis of the results of the immunisation experiment showed a directrelationship of the immunological response to the DNA dose. The resultsalso suggest that the plasmid containing the chIL-2 expression cassettestimulates the immunological response (the anti-HA antibodies level)only when low doses of K3 plasmid were applied. This is in agreementwith the dot blot results.

Example V Analysis of the Immunological Response by theHaemagglutination Inhibition (HI) Test

In order to detect antibodies able to inhibit the haemagglutinationprocess, an HI test was performed. The HI test is based on the affinityof antibodies in the sera of immunised chickens to the H5 antigen of lowpathogenicity strain H5N2. The homology of the amino acid sequence ofthe strain used and the vaccine antigen was 91% (data not shown). The HItest was conducted according to the standard procedure (Avian Influenzain: OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals,2010). Samples (250 of the serial two-fold dilutions (1:8 to 1:512) ofthe sera were incubated with 4 units of HA of the heterologousinactivated H5N2 antigen (GD Deventer, Netherlands) in titration platesat room temperature. After 25 min. of incubation, 25 μl of 1% henerythrocytes was added and incubated for 30 min. The reciprocal of thehighest dilution which inhibits agglutination of the hen's erythrocytesdenotes the value of the HI of the examined sera. Samples were assignedas positive at a titer value≧8.

The titers of particular hens were shown as log₂.

No antibodies inhibiting haemagglutination were detected in any group ofchickens after the first collection of blood (day 18). In the seracollected on day 33, neutralising antibodies were detected in the groupsvaccinated with the K3 plasmid. The best results were observed amongchickens immunised with the highest dose of K3 (FIG. 3; B1 group).

Example VI Comparison of the Immunological Response on the DNA VaccineContaining Optimised and Non-Optimised cDNA

Two groups of chickens were immunised twice on day 6 and on day 21 oflife. The vaccines were administered subcutaneous. The first group wasimmunised with 250 μg of K3 plasmid carrying modified cDNA (SEQ. 2). Thesecond one was immunised with 250 μg of the non-optimised codingsequence (SEQ. 1). Sera collected on day 42 were used to evaluate thespecific anti-HA antibodies. The immunological response was evaluated bythe technique of the dot blot and HI tests described in Example III. Theresults showed that the vaccine containing modified cDNA was moreefficient than the non-modified one (FIG. 4).

Example VII Comparison of Two Routes of Vaccine Administration:Subcutaneous and Intramuscular

Chickens were immunised according to scheme B. 7-day old broilerchickens were divided into 11 groups, immunised twice 2 weeks apart, asindicated in scheme B, and slaughtered 3 weeks following the secondimmunisation. The birds were immunised intramuscularly in the breastmuscle or subcutaneously in the neck with 250, 125 or 62 μg of DNA in afinal volume of 320, 160 or 100 μl, respectively. Two groups receivedonly one dose of the experimental vaccine, and two groups received plainDNA without the liposomal carrier (scheme B). Blood samples werecollected twice, on day 20—before boost immunisation, and on day 42—3weeks after the second (boosting) immunisation. Sera were prepared asdescribed in Example II. The evaluation of the immunological response tothe DNA vaccination was conducted by ELISA assay and the HI test exactlyas described in Example IV and V. On day 20, when blood samples werecollected for the first time (two weeks after the priming immunisation),the response was still undeveloped, and antibody titers were low (FIG.5). After the boosting immunisation, the titers increased sharply, andsome of the tested groups presented strong positive results, includingthe groups immunised with 250 μg and 125 μg administered subcutaneouslyor with 125 μg of plasmid DNA administered intramuscularly. The worstresults were obtained in the group without the liposomal carrierinjected subcutaneously and in a group where individuals received 60 μgof DNA also injected subcutaneously. A summary of these results islisted in Table 1.

TABLE 1 Results of immunisation experiments % positive Results Resultsper group Group (day 20) (day 42) (day 42) No. Immunisation ELISA ELISAHI ELISA HI 1 **sc250 1/7  4/7 0/7 57% 0% 2 sc250 1/12 11/12  5/12 92%42% 3 sc125 3/15 13/15  8/15 87% 53% 4 sc62 0/7  3/7 0/7 43% 0% 5 *sc2500/8  5/8 1/8 63% 12% 6 ***sc250 0/7  3/7 0/7 43% 0% 7 im125 9/12 12/1210/12 100% 83% 8 im62 5/12 10/12  7/12 83% 58% 9 *im125 9/12 10/12 83%10 ***im125 3/12 12/12 100% 11 pCI vector 0/10  0/10  0/10 0% 0%sc—subcutaneous, im—intramuscular, *group received one dose, **wild typeHA sequence, ***immunisation without liposomal carrier

Scheme B of subcutaneous and intramuscular chicken immunisation. PrimingBoosting immunisation immunisation (2 weeks after priming) No. of GroupDosage Dosage chickens in a No. Formulation (μg) Formulation (μg) group1 HA sc 250 HA opt sc 250 7 2 HA opt sc 250 HA opt sc 250 12 3 HA opt sc125 HA opt sc 125 15 4 HA opt sc 62 HA opt sc 62 7 5 — — HA opt sc 250 86 HA opt sc-L 250 HA opt sc-L 250 7 7 HA opt im 125 HA opt im 125 12 8HA opt im 62 HA opt im 62 12 9 HA opt im 125 — — 12 10 HA opt im-L 125HA opt im-L 125 12 11 pCI vector 125 pCI vector 125 10HA—haemagglutinine construct, HAopt—haemagglutinin optimised construct,sc—subcutaneous, im—intramuscular, -L—without a liposomal carrier

The results obtained in the haemagglutination inhibition test were lessobvious. Nevertheless, anti-HA antibodies at a level associated withprotection against the influenza virus were detected (FIG. 6). After thepriming immunisation, there were no detectable responses (data notshown), although 3 weeks after boosting, the immune response rose. Thehighest titers of antibodies were detected in the group vaccinatedintramuscularly with 125 μg of DNA, where the mean titer (log₂) wasgreater than 4, which is usually taken as a protective level. Nopositive results were found in groups immunised with wild type(non-optimised codones) HA, immunised without liposomal carrier andimmunised subcutaneously with 60 μg of the plasmid DNA.

Based on those two assays (ELISA and HI), the intramuscular route of twodoses of experimental vaccine was considered to be the most promisingscheme and was chosen for the challenge experiment.

Example VIII Challenge Experiment

The HPAI/H5N1/turkey/35/07 and A/crested eagle/Belgium/01/04 influenzavirus strains were cultivated in the allantoic cavities of embryonatedchicken eggs in the National Veterinary Research Institute in biosafetylevel 3 conditions. SPF chickens were immunised intramuscularly twicewith 125 μg of plasmid DNA as previously described (Example VI). Thecontrol chickens remained untreated. 3 or 8 weeks after the secondimmunisation, the animals were inoculated intranasally (conjunctively)with 10⁶EID₅₀ of the virus. 24 hours after inoculation, one or twountreated chickens were added to the group of vaccinated and infectedchickens to monitor virus transmission. Scheme C shows the details ofthis experiment. The birds were monitored daily for 14 days for signs ofdisease. Tracheal and cloacal swabs were collected on day 3, 7 and 10p.i. in order to monitor virus proliferation. The animals wereslaughtered 2 weeks after the challenge. Samples of several organs weretaken from the birds that died during the observation.

Scheme C of challenge experiment of SPF chickens No. of Priming Boostingchickens Group immunisation immunisation Virus infection in No. day dayday virus a group 1 7 21 42 A/tk/35/07 10 (+2) 2 7 21 77 A/tk/35/07  5(+1) 3 — — 42 A/tk/35/07 5 4 7 21 42 A/ce/01/ 10 (+2) 04 5 — — 42A/ce/01/ 5 04

All groups of control chickens showed replication of the influenza virusat high titers. Moreover, these chickens showed signs of disease ofpathogenic AI and died 2 or 3 days after infection. The immunised groupchallenged with the homologues strain had no signs of infection and mosthad no virus replication. All birds in this group survived. In the groupchallenged 3 weeks after the second vaccination, two transmissioncontrol chickens also survived the challenge clinically healthy. Theimmunised chickens were completely protected from the lethal challengewithout any clinical signs and virus shedding. In the group challenged 8weeks after the second vaccination, one of two control chickens haddetectable virus replication and died on day 7 after infection (FIGS. 7Aand 7B).

In the group challenged with the heterologous strain, the majority ofchickens had no signs of infection and no virus replication; however, 3birds died at 5, 10 and 13 days after infection. The last one had nodetectable virus replication in swabs and organs. The survival ratio was70%. Two transmission control chickens had detectable virus replicationand died on day 3 and 4 p.i. (FIG. 7C).

REFERENCES

-   -   Avian Influenza in: OIE Manual of Diagnostic Tests and Vaccines        for Terrestrial Animals 2010        [http://www.oie.int/international-standard-setting/terrestrial-manual/access-online/]    -   Dhama, K., Mahendran, M., Gupta, P. K. and Rai, A. (2008). DNA        vaccines and their applications in veterinary practice: current        perspectives. Vet Res Commun 32, 341-356.    -   Fynan, E. F., Robinson, H. L., and Webster, R. G. (1993a). Use        of DNA encoding influenza haemagglutinin as an avian influenza        vaccine. DNA Cell Biol 12, 785-789.    -   Fynan, E. F., Webster, R. G., Fuller, D. H., Haynes, J. R.,        Santoro, J. C. and Robinson, H. L. (1993b). DNA vaccines:        protective immunizations by parenteral, mucosal and gene-gun        inoculations. Proc Natl Acad Sci USA 90, 11478-11482.    -   Gromadzka, B., Smietanka, K., Dragun, J., Minta, Z.,        Gora-Sochacka, A. and Szewczyk, B. (2008). Detection of changes        in avian influenza genome fragments by multitemperature        single-strand conformational polymorphism. Mol Cell Probes 22,        301-304.    -   Kodihalli, S., Kobasa, D. L. and Webster, R. G. (2000).        Strategies for inducing protection against avian influenza A        virus subtypes with DNA vaccines. Vaccine 18, 2592-2599.    -   Kutzler, M. A. and Weiner, D. B. (2008) DNA vaccines: ready for        prime time? Nat Rev Genet 9, 776-788.    -   Park, J. H., Sung, H. W., Yoon, B. I. and Kwon, H. M. (2009).        Protection of chicken against very virulent IBDV provided by in        ovo priming with DNA vaccine and boosting with killed vaccine        and the adjuvant effects of plasmid-encoded chicken        interleukin-2 and interferon-gamma. J Vet Sci 10, 131-139.    -   Rao, S., Kong, W. P., Wei, C. J., Yang, Z. Y., Nason, M.,        Styles, D., DeTolla, L. J., Panda, A., Sorrell, E. M., Song, H.,        Wan, H., Ramirez-Nieto, G. C., Perez, D. and Nabel, G. J.        (2008). Multivalent HA DNA vaccination protects against highly        pathogenic H5N1 avian influenza infection in chickens and mice.        PLoS One 3, e2432.    -   Robinson, H. L., Hunt, L. A., and Webster, R. G. (1993).        Protection against a lethal influenza virus challenge by        immunization with a haemagglutinin-expressing plasmid DNA.        Vaccine 11, 957-960.    -   Swayne, D. E. (2009). Avian influenza vaccines and therapies for        poultry. Comp Immunol Microbiol Infect Dis 32, 351-363.

1. DNA vaccine containing the modified cDNA encoding the H5haemagglutinin protein, characterised in that, it contains the deletionof the region encoding the amino acids at the proteolytic cleavagebetween subunits HA1 and HA2 and codons altered for codons optimal forchickens in order to ensure maximal yield of the H5 protein in birdcells.
 2. DNA vaccine according to claim 1, characterised in that, itcontains the sequence defined as SEQ. ID No.
 2. 3. DNA vaccine accordingto claim 1, characterised in that the deletion includes between 15 and21 base pairs encoding the amino acids at the site of proteolyticcleavage between subunits HA1 and HA2, preferably 18 base pairs. 4.Method of induction of an immunological response of the humoral type inthat specific antibodies recognising the H5 haemagglutinin protein areproduced in immunised poultry, characterised in that, the DNA vaccinedescribed in claim 1 is applied.
 5. Method according to claim 4,characterised in that, the immunological response is enhanced byadministration of the cDNA of chicken interleukin 2, defined as SEQ. IDNo.
 3. 6. Method according claim 4, characterised in that, the firstdose of the vaccine is administered up to 14 days after hatching. 7.Method according to claim 4, characterised in that, the DNA suspensionis prepared and the vaccine samples for immunisation containing a DNAsuspension in a lipid or macromolecular carrier are prepared.
 8. Methodof immunisation, characterised in that, at least one dose of the DNAvaccine described in claim 1 is applied.
 9. Method according to claim 8,characterised in that, the first dose of the vaccine is DNA vaccine andthe second dose of the vaccine is the DNA vaccine or the antigenicprotein, HA.
 10. Antibodies specifically recognising the H5haemagglutinin protein of the influenza virus, characterised in thatthey were obtained by use of the vaccine according to claim
 1. 11.Antibodies according to claim 9, characterised in that, they are activeor inactive in the haemagglutinin inhibition test performed with the H5antigen of the influenza virus.
 12. Antibodies according to claim 9,characterised in that, they are able or not able to neutralise theinfluenza virus.
 13. A method comprising: using the DNA vaccinedescribed in claim 1, in order to obtain antibodies specificallyrecognising the H5 haemagglutinin protein of the influenza virus.